JP4169344B2 - Alkaline phosphatase stabilization method - Google Patents
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Abstract
Description
技術分野
本発明は、臨床検査用アルカリホスファターゼ含有凍結乾燥製剤に関する。更に詳しくは、凍結乾燥製剤でのヒト由来アルカリホスファターゼの安定化方法、より具体的には、復水後の活性上昇が認められず、かつ、長期保存が可能な凍結乾燥製剤及びその安定化方法に関する。
背景技術
アルカリホスファターゼ(ALP)は、植物、動物、及び微生物などに存在することが知られている酵素である。例えば、動物起源としては、牛小腸、ブタ小腸、ウサギ小腸、犬小腸、牛腎臓、ブタ腎臓、そしてヒト胎盤など多岐にわたり、また細菌起源として、大腸菌由来の酵素などがあり、これらは市販されている。これらの酵素は、作用という点から見れば同一であるが、特異性、熱安定性、反応性などの諸性質は異なっている。
臨床検査においては、種々の病態を反映するヒト血清中の酵素活性が測定されている。血清中に出てくる酵素の起源としては、肝臓、腎臓、骨、小腸、胎盤など多岐にわたっている。
一般に酵素活性は、pHや測定温度などの条件によって変動する相対的な数値として表されるため、同じサンプルを用いた場合でも使用する試薬により活性値は異なる。また、試薬の劣化や検体自体の劣化などの要因も活性値へ大きく影響を与える。従って、酵素活性を精密に測定するには、測定のたびに安定で変動のない基準物質を用いて、検体の測定結果と比較するか、測定方法を統一するかしなければならない。
臨床検査分野では、酵素活性に関する基準物質は、その目的に応じて、管理血清、キャリブレーター、標準物質などという慣用名で市販品が流通している。このうち、管理血清は、内部精度管理、すなわち、ある測定施設での日常の管理に、また、キャリブレーター、標準物質は、精度管理はもとより、測定された値の正確さまで加味され、多施設間での管理物質として用いられている。
しかしながら、ヒトの血清の中でのALP酵素活性は変動しやすく、新鮮血で96時間後の活性の変動率は保存条件によって、−4%〜10%であり、また、凍結プール血清を溶解後、室温で保存した場合は6.4%活性が上昇したと報告されている(Clin.Chem.,vol.18(4),1972)。また、熱安定性はアイソザイムの種類により異なることが知られている(玄番昭夫、アイソエンザイム、医学書院、p10−16、1978)。
一方、凍結乾燥は、蛋白質などのように、熱に対して不安定であり、水溶液として放置しておくと変性しやすい物質の長期保存法として有用であり、汎用されている。酵素も、その活性を長期に保つために凍結乾燥されることが多い。しかし、酵素の種類、純度などから、凍結乾燥の過程で、変性、失活してしまうものも少なくない。そのため、目的物質の他にアルブミンなどの蛋白質や、ショ糖、トレハロースなどの糖を安定化剤として添加し、この問題が回避されてきた。例えば、特開昭56−148291号公報には、ショ糖とウシまたはヒト血清アルブミンを含む酵素の安定化剤についての記載がある。しかし、安定化剤として全ての蛋白質に効果が認められるようなものはなく、各蛋白毎に有効な安定化剤が個別に検討されているのが実状である。
精度管理用やその他の目的のために、ALPの凍結乾燥に関し種々報告されている。例えば、凍結乾燥に及ぼすALPに対する糖質添加効果について、トレハロース、マンニトール、ラクトースが比較されている(J.Pharm.Pharmacol.,45(10),86−93,1993)。これによると、安定化効果の大きさはトレハロース、ラクトース、マンニトールの順であるが、用いられている酵素は、牛小腸粘膜由来の粗精製酵素のみであり、ヒト由来酵素の検討はなされていない。また、J.Pharm.Pharmacol.,45(10),900−906,1993には、やはり牛小腸粘膜由来の精製酵素を用い、添加剤として、糖化されたヒト血清アルブミンを用いて凍結乾燥を実施すると、マイナスの効果を与えることが示されている。糖化されたアルブミンは、還元糖とアルブミンとの組合せで生じるので、この組合せを添加剤として用いるのは好ましくないとされている。また、比較例として、15%のトレハロースのみを添加剤として用いると、凍結乾燥の過程で活性が失われ、乾燥後に約40%の残存活性しか示さないことが、報告されている。
先に述べたように、基準物質としてまず備えていなければならないのは、その物質が保証された期間、一般的には1年間以上の間に安定であり、かつ、使用時に一定の性能を示すことである。従来より、市販されている凍結乾燥された管理血清を溶解するとALPは、徐々に活性が上昇するとの指摘がなされており(Clin.Chem.vol.18(4),p366−377,1972)、これは溶解後に使用する場合に、決して好ましいとはいえない。また、ヒト血清を凍結したものと、凍結乾燥したものとの両者の精度管理物質の比較では、特にALP及びクレアチンキナーゼ(CK)の場合、凍結したものの方が管理血清としては優れているとの報告もある(Clin.Biochem.,29(2),183−185,1996)。これらの管理血清は、内在するヒト由来のALPの他に動物起源の酵素を血清に加えて調製されているものが多い(Cli.Chem.,35(3),p510,1989)。本来、ヒト由来酵素を使用すべきであるのに、性質の異なる動物起源の酵素が多々用いられてきたのは、倫理的な観点、感染、取得し易さなどからであったが(Clin.Chem.,33(11),p1971−1977,1987)、近年は、生体材料を用いずに、株化動物細胞や遺伝子組換え技術による形質転換細胞から取り出すことも容易になり、ヒト由来酵素を用いた商品も入手できるようになった。例えば、ヒト羊膜株化細胞より取得されたALPを含む凍結乾燥された多酵素からなる管理血清の場合も、溶解後の活性上昇が認められる(生物試料分析、14(2),p81−89,1991:臨床検査・機器・試薬、15(4),p615−623,1992)。
このように、今後ますますヒト由来ALPを用いた精度管理物質の開発が加速されつつある状況であるが、いまだヒト由来ALPを用い、復水後に活性の上昇が認められず、長期的に安定な凍結乾燥物は報告されていない。
本発明の目的は、ヒト由来ALPを用い、復水後に活性の上昇が認められず、長期的にも安定な凍結乾燥された製剤及びその製剤におけるヒト由来ALPの安定化方法を提供することである。
発明の開示
本発明者は、上記課題を解決するために鋭意検討した結果、ヒト由来ALPの凍結乾燥時に、ガラクトース、ラクトース、及びフルクトースからなる群より選ばれる糖と、アルブミンあるいはデキストランとを添加すると、意外にも凍結乾燥前後で活性の変動が低く、かつ長期的に安定な組成物が得られることを見いだし、本発明を完成した。すなわち、本発明によれば、ヒト由来ALPをガラクトース、ラクトース、及びフルクトースからなる群より選ばれる糖と、アルブミンあるいはデキストランの存在のもとに凍結乾燥することにより、長期保存可能で、かつ、使用時すなわち溶解時に活性上昇等の変動のない実用に優れたヒト由来ALPの安定化製剤が提供される。
本発明に関して、以下具体的に説明する。本発明の凍結乾燥製剤に用いることのできる糖としては、ガラクトース、ラクトース、フルクトースが好ましく、糖の濃度としては0.5〜20(W/V)%、特に1〜10(W/V)%が好ましい。これらの糖は単独でも2種あるいは3種の混合物として用いることもできる。
また、アルブミンとしては、例えばヒトや牛の血清アルブミン(BSA)をはじめとした哺乳動物、また、ニワトリ血清アルブミン等の鳥類等のアルブミンを使用することができ、その濃度として好ましくは0.3〜7(W/V)%、特に1〜6(W/V)%が好ましい。更に、これら各種アルブミンのアミノ酸をコードする遺伝子が組み込まれた形質転換細胞の培養により取得、精製されたものも使用することもできる。アルブミンは賦型剤として用いるのであるが、アルブミンと同等の効果を示す他の蛋白質、デキストランなどの多糖類なども、単独あるいは組み合わせて適宜用いることもできる。デキストランの使用量としては、0.3〜7(W/V)%、特に1〜5(W/V)%が好ましい。
また、本発明に用いることのできアルカリホスファターゼとしては、ヒト由来酵素が好ましい。ヒト由来ALPの起源としては、肝臓、腎臓、骨、小腸、胎盤が知られており、これら生体材料から酵素が取得できる。胎盤由来酵素は、市販されており入手可能であるが、これら生体材料からの取得は、倫理的観点や、感染性などからあまり好ましいとはいえない。また、HeLa細胞や羊膜細胞などの株化細胞の培養物から取得することもできる。更に、近年の遺伝子工学技術の発展により、ヒト由来ALP蛋白質をコードする遺伝子も明らかになっており、これらの遺伝子を組み込んだ形質転換体細胞からも取得できる。例えば、肝型ALPが、形質転換動物細胞より取得、精製され、旭化成(株)より市販されている(旭化成(株)診断用酵素カタログ)。
ここで、ヒト肝型ALP遺伝子は、ヒト臓器非特異的ALP遺伝子と言われているものと同一である。また、この際、形質転換体細胞としては、ヒト細胞のみならず、チャイニーズハムスター由来CHO細胞等のヒト以外の動物細胞や大腸菌、酵母、カビなどの微生物を用いることもできる。更にALPのアミノ酸配列の一部が削除または置換され、他のアミノ酸残基またはアミノ酸配列が付加されているALP誘導体をコードする遺伝子を用いた形質転換体を用いることもできる。これら生産された酵素は、市販されたもの以外は、カラムクロマトグラフィ法等の公知の精製法を組み合わせ、実用レベルまで純度を高めて本目的に使用すればよい。
本発明の凍結乾燥製剤におけるALPの添加量は、特に限定されないが、9〜6500U/L、特に好ましくは45〜1300U/Lである。
また、凍結乾燥前の水溶液のpHは中性付近、特に6.5〜8.5付近であり、凍結乾燥品を溶解させた時のpHも同等であることが望ましいので、適当な緩衝剤、例えばPIPES、HEPES、BES等のグッドの緩衝液や、リン酸緩衝液、トリス緩衝液などを5〜200mM、特に10〜100mMの濃度で使用すればよい。また、凍結乾燥の形状を良好にするために用いられる各種添加物、例えばデキストランやデキストラン硫酸、マンニトール等の糖アルコールなども適宜用いることができる。
また、ALPは亜鉛をその分子中にもつことが知られているので、例えば塩化亜鉛を添加したり、また活性化剤として知られている塩化マグネシウムを適宜添加することもできる。また、酵素の安定化効果が知られているアミノ酸、例えばバリン等を適宜添加してもよい。
発明を実施するための最良の形態
本発明を実施例に基づいて説明する。
(実施例1)
5%ラクトース、0.5mM塩化マグネシウム、10μM塩化亜鉛、400U/Lヒト肝由来ALP(旭化成(株)製品ナンバーT−73)を含む20mMPOPSO−NaOH緩衝液(pH7.5)に各々BSAを(1)無添加、(2)1(W/V)%、(3)3(W/V)%、になるように加え、2mlずつバイアル瓶に分注し、凍結乾燥を実施した。各々の条件につき、37℃の負荷試験を行い1週間毎、4週間まで、測定時に各1本を2mlの蒸留水で溶解し、ALP活性を測定した。残存活性の推移を表1に示す。BSA無添加の系に比べて、BSAが添加された系で有意に安定化が認められた。
(実施例2)
3%BSA、0.1mM塩化亜鉛、30mMバリン、400U/Lヒト肝由来ALPを含む20mMBES−NaOH緩衝液(pH7.5)に各々3%のシュークロース、ガラクトース、ラクトースを加えて、3mlずつ各々2本バイアル瓶に分注し、凍結乾燥を実施した。凍結乾燥品に3mlの蒸留水を加えて溶解し、ALP活性を測定した。残りの1本を37℃の恒温器に入れて1週間放置した。その後同様にALP活性を測定した。表2に示したように、本発明のガラクトース、ラクトースが添加されたものは、シュークロース添加系に比べて残存活性が有意に高かった。
(実施例3)
3%BSA、0.5mM塩化マグネシウム、10μM塩化亜鉛、400U/Lヒト肝由来ALP(旭化成(株)製品ナンバーT−73)を含む40mMPIPES−NaOH緩衝液に各々5(W/V)%のガラクトース、フルクトース、ラクトースを加え、2mlずつバイアル瓶に分注し、凍結乾燥を実施した。凍結乾燥の条件は、凍結温度−50℃、真空にしたのち、一次乾燥−10℃、12時間、二次乾燥20℃、24時間で実施し、真空打栓した。凍結乾燥品に、2mlの蒸留水を加えて、溶解し、各々の糖添加物につきそれぞれALP活性を測定した。残りのバイアル瓶を37℃の恒温器に入れて、1週間毎に1本ずつ2mlの蒸留水にて溶解させ、3週間までALPの活性を測定した。凍結乾燥直後の酵素活性を100%として残存活性を表3に示した。糖無添加に比べて、ガラクトース、フルクトース、ラクトースいずれの糖においても残存活性は有意に高かった。
(実施例4)
3%BSA、0.5mM塩化マグネシウム、10μM塩化亜鉛、400U/Lヒト肝由来ALP(旭化成(株)製品ナンバーT−73)を含む40mMBES−NaOH緩衝液に各々(1)糖無添加、(2)ガラクトース1(W/V)%、(3)3(W/V)%、(4)5(W/V)%、(5)マンノース3(W/V)%を加え、2mlずつバイアル瓶に分注し、凍結乾燥を実施した。各々の条件につき、37℃の負荷試験を行い1週間毎、4週間まで、測定時に各1本を2mlの蒸留水で溶解し、ALP活性を測定した。残存活性の推移を表4に示す。ガラクトース添加の効果が認められたが、マンノースは3週間目以降急激に活性が低下した。
(実施例5)
1%BSA、3%デキストラン60K、0.5mM塩化マグネシウム、10μM塩化亜鉛、400U/Lヒト肝由来ALP(旭化成(株)製品ナンバーT−73)を含む40mMBES−NaOH緩衝液に各々ガラクトース5(W/V)%、または3ラクトース5(W/V)%を加え、2mlずつバイアル瓶に分注し、凍結乾燥を実施した。凍結乾燥品を溶解し、初期の活性を測定したのち、それぞれ4本ずつ、25℃、37℃、45℃の恒温器に入れ、負荷試験を実施した。結果を表5に示す。次に、この結果を基に、アレニウス(Arrhenius)式を用いて、4℃及び−10℃保存の場合に適用した(J.Biol.Stand.,12,p195−224,1984)。残存活性が98%になるまでの期間を計算し、表6に示した。ガラクトース、ラクトースいずれの場合も4℃保存で1年以上、−10℃保存では、約15年と長期にわたり安定であることが予測される計算結果であった。
(実施例6)
3%BSA、0.5mM塩化マグネシウム、10μM塩化亜鉛、400U/Lヒト肝由来ALP(旭化成(株)製品ナンバーT−73)を含む40mMBES−NaOH緩衝液に各々5(W/V)%のガラクトースまたはラクトースを、比較例としてトレハロースを加えて、バイアルに2ml分注し、凍結乾燥した。冷蔵(5℃)での活性変動を6ヶ月間調べて表7に示した。トレハロース添加の凍結乾燥製剤が、4ヶ月で8.6%活性低下が認められるのに対して、本発明の製剤では、活性低下は認められない。
(実施例7)
3%BSA、0.5mM塩化マグネシウム、10μM塩化亜鉛、400U/Lヒト肝由来ALP(旭化成(株)製品ナンバーT−73)を含む40mMBES−NaOH緩衝液に各々5(W/V)%のガラクトース、ラクトース、フルクトースを加えて、2mlずつバイアルに分注して凍結乾燥した。2mlの蒸留水で溶解し、25℃に放置した。溶解液中のALP活性の推移を残存活性として表8に表した。いずれの糖においても活性上昇は認められず、48時間まで安定であった。
(実施例8)
5%ガラクトス、0.01mM塩化亜鉛、0.5mM塩化マグネシウム、400U/lヒト肝由来ALPを含む20mMPOPSO−NaOH緩衝液(pH7.5)に各々3%のデキストラン(分子量60000−90000:和光純薬工業(株))、あるいは3%のBSA、を加えて、2mlずつバイアル瓶に分注し、実施例3と同様の凍結乾燥条件にて凍結乾燥を実施した。各々の条件につき、37℃の負荷試験を行い、1週間毎、4週間まで、測定時に各1本を2mlの蒸留水で溶解し、ALP活性を測定した。残存活性の推移を表9に示す。デキストランにおいてもBSAに比べ若干劣るものの安定化効果が確認できた。尚、デキストラン、BSAともに無添加のものを対照に置いたが、うまく凍結乾燥できなかった。
産業上の利用可能性
本発明によれば、臨床検査用アルカリホスファターゼ含有凍結乾燥製剤として、具体的には、復水後の活性上昇が認められず、かつ長期保存が可能なヒト由来アルカリホスファターゼの安定な凍結乾燥製剤を提供できる。 TECHNICAL FIELD The present invention relates to a lyophilized preparation containing alkaline phosphatase for clinical testing. More specifically, a method for stabilizing human-derived alkaline phosphatase in a freeze-dried preparation, more specifically, a freeze-dried preparation that does not show increased activity after condensate and can be stored for a long period of time, and a method for stabilizing the same About.
BACKGROUND ART Alkaline phosphatase (ALP) is an enzyme known to exist in plants, animals, microorganisms and the like. For example, there are a wide variety of animal origins such as cattle small intestine, pig small intestine, rabbit small intestine, dog small intestine, cow kidney, pig kidney, and human placenta, and bacterial origin includes E. coli-derived enzymes, which are commercially available. Yes. These enzymes are the same in terms of action, but have different properties such as specificity, thermal stability, and reactivity.
In clinical tests, enzyme activities in human serum that reflect various pathological conditions are measured. The sources of enzymes appearing in serum are diverse, such as liver, kidney, bone, small intestine, and placenta.
In general, enzyme activity is expressed as a relative numerical value that varies depending on conditions such as pH and measurement temperature. Therefore, even when the same sample is used, the activity value varies depending on the reagent used. In addition, factors such as reagent degradation and specimen degradation also greatly affect the activity value. Therefore, in order to accurately measure the enzyme activity, it is necessary to use a reference material that is stable and does not fluctuate for each measurement, and to compare with the measurement result of the specimen or to unify the measurement method.
In the clinical laboratory field, as for reference substances relating to enzyme activity, commercially available products such as controlled sera, calibrators and standard substances are distributed according to their purposes. Of these, the control serum is used for internal quality control, that is, daily management at a certain measurement facility, and the calibrator and reference material are not only controlled for accuracy but also to the accuracy of the measured value. It is used as a controlled substance.
However, the ALP enzyme activity in human serum is likely to fluctuate, and the rate of activity change after 96 hours in fresh blood is -4% to 10% depending on the storage conditions. It has been reported that the activity increased by 6.4% when stored at room temperature (Clin. Chem., Vol. 18 (4), 1972). Moreover, it is known that thermal stability changes with kinds of isozymes (Akio Genban, Isoenzyme, Medical School, p10-16, 1978).
On the other hand, lyophilization is useful as a long-term storage method for substances such as proteins that are unstable to heat and easily denatured when left as an aqueous solution. Enzymes are also often lyophilized to keep their activity for a long time. However, due to the type and purity of the enzyme, there are many things that are denatured and inactivated during the freeze-drying process. Therefore, in addition to the target substance, proteins such as albumin and sugars such as sucrose and trehalose are added as stabilizers to avoid this problem. For example, Japanese Patent Laid-Open No. 56-148291 describes an enzyme stabilizer containing sucrose and bovine or human serum albumin. However, there are no stabilizers that are effective for all proteins, and it is the actual situation that effective stabilizers are individually examined for each protein.
Various reports on freeze-drying of ALP have been reported for quality control and other purposes. For example, trehalose, mannitol, and lactose have been compared for the effect of carbohydrate addition to ALP on lyophilization (J. Pharm. Pharmacol., 45 (10), 86-93, 1993). According to this, the stabilizing effect is in the order of trehalose, lactose, and mannitol, but the only enzyme used is a crudely purified enzyme derived from bovine small intestinal mucosa, and no human-derived enzyme has been studied. . In addition, J.H. Pharm. Pharmacol. , 45 (10), 900-906, 1993, a purified enzyme derived from bovine small intestinal mucosa is used, and freeze-drying using glycated human serum albumin as an additive gives a negative effect. It is shown. Since saccharified albumin is produced by a combination of reducing sugar and albumin, it is not preferable to use this combination as an additive. As a comparative example, it has been reported that when only 15% trehalose is used as an additive, the activity is lost during the freeze-drying process and only about 40% residual activity is exhibited after drying.
As mentioned above, the first thing to have as a reference material is that the material is stable for a guaranteed period of time, typically over a year, and exhibits a certain performance when used. That is. Conventionally, it has been pointed out that ALP gradually increases in activity when commercially available lyophilized control serum is dissolved (Clin. Chem. Vol. 18 (4), p366-377, 1972). This is never preferred when used after dissolution. In addition, when comparing the quality control substances of human serum frozen and lyophilized, it was found that the frozen serum was superior as the control serum especially in the case of ALP and creatine kinase (CK). There is also a report (Clin. Biochem., 29 (2), 183-185, 1996). Many of these control sera are prepared by adding an animal-derived enzyme to the serum in addition to the endogenous human-derived ALP (Cli. Chem., 35 (3), p510, 1989). Originally, human-derived enzymes should be used, but many animal-derived enzymes having different properties have been used from an ethical point of view, infection, and ease of acquisition (Clin. Chem., 33 (11), p1971-1977, 1987), in recent years, it has also become easy to take out from an established animal cell or a transformed cell by gene recombination technology without using a biomaterial, The products used are now available. For example, in the case of a control serum consisting of a lyophilized multienzyme containing ALP obtained from a human amnion cell line, an increase in activity after lysis is observed (biological sample analysis, 14 (2), p81-89, 1991: Clinical examination / equipment / reagent, 15 (4), p615-623, 1992).
In this way, the development of quality control substances using human-derived ALP is accelerating in the future. However, human-derived ALP is still used, and there is no increase in activity after condensing, and it is stable over the long term. No lyophilized product has been reported.
An object of the present invention is to provide a lyophilized preparation that uses human-derived ALP, has no increase in activity after condensing, and is stable over the long term, and a method for stabilizing human-derived ALP in the preparation. is there.
DISCLOSURE OF THE INVENTION As a result of intensive studies to solve the above-mentioned problems, the present inventor has found that a saccharide selected from the group consisting of galactose, lactose, and fructose, and albumin or dextran when lyophilizing human-derived ALP. Surprisingly, it was found that a composition having a low activity fluctuation before and after lyophilization and stable over the long term was obtained, and the present invention was completed. That is, according to the present invention, human-derived ALP can be stored for a long period of time by lyophilization in the presence of a sugar selected from the group consisting of galactose, lactose, and fructose, and albumin or dextran. A stabilized preparation of human-derived ALP which is excellent in practical use without fluctuations such as increased activity at the time of dissolution is provided.
The present invention will be specifically described below. The sugar that can be used in the freeze-dried preparation of the present invention is preferably galactose, lactose, or fructose, and the sugar concentration is 0.5 to 20 (W / V)%, particularly 1 to 10 (W / V)%. Is preferred. These sugars can be used alone or as a mixture of two or three kinds.
Further, as albumin, albumin such as mammals including human and bovine serum albumin (BSA), and birds such as chicken serum albumin can be used, and the concentration is preferably 0.3 to 7 (W / V)%, particularly 1 to 6 (W / V)% is preferable. Furthermore, those obtained and purified by culturing transformed cells into which genes encoding amino acids of these various albumins have been incorporated can also be used. Albumin is used as an excipient, but other proteins exhibiting the same effect as albumin, polysaccharides such as dextran, etc. can be used appropriately alone or in combination. The amount of dextran used is preferably 0.3 to 7 (W / V)%, particularly 1 to 5 (W / V)%.
Moreover, as an alkaline phosphatase that can be used in the present invention, a human-derived enzyme is preferable. As the origin of human-derived ALP, liver, kidney, bone, small intestine and placenta are known, and enzymes can be obtained from these biomaterials. Placenta-derived enzymes are commercially available and available, but acquisition from these biomaterials is not very preferable from an ethical point of view or infectivity. It can also be obtained from a culture of established cells such as HeLa cells and amniotic cells. Furthermore, with the recent development of genetic engineering technology, genes encoding human-derived ALP proteins have also been clarified and can be obtained from transformed cells incorporating these genes. For example, liver-type ALP is obtained and purified from transformed animal cells and is commercially available from Asahi Kasei Corporation (Asahi Kasei Corporation diagnostic enzyme catalog).
Here, the human liver-type ALP gene is the same as the so-called human organ non-specific ALP gene. At this time, not only human cells but also non-human animal cells such as Chinese hamster-derived CHO cells and microorganisms such as Escherichia coli, yeast, and mold can be used as transformant cells. Furthermore, a transformant using a gene encoding an ALP derivative in which a part of the amino acid sequence of ALP is deleted or substituted and another amino acid residue or amino acid sequence is added can also be used. These produced enzymes may be used for this purpose after being combined with a known purification method such as a column chromatography method to increase the purity to a practical level.
The amount of ALP added in the freeze-dried preparation of the present invention is not particularly limited, but is 9-6500 U / L, particularly preferably 45-1300 U / L.
In addition, since the pH of the aqueous solution before lyophilization is near neutral, particularly around 6.5 to 8.5, and it is desirable that the pH when the lyophilized product is dissolved is the same, an appropriate buffer, For example, Good's buffer such as PIPES, HEPES, and BES, phosphate buffer, Tris buffer, etc. may be used at a concentration of 5 to 200 mM, particularly 10 to 100 mM. Various additives used for improving the freeze-dried shape, for example, sugar alcohols such as dextran, dextran sulfate, and mannitol can be used as appropriate.
Further, since ALP is known to have zinc in its molecule, for example, zinc chloride can be added, or magnesium chloride known as an activator can be added as appropriate. An amino acid known to have an enzyme stabilizing effect, such as valine, may be added as appropriate.
BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described based on examples.
(Example 1)
Each BSA was added to a 20 mM POPSO-NaOH buffer solution (pH 7.5) containing 5% lactose, 0.5 mM magnesium chloride, 10 μM zinc chloride, 400 U / L ALP derived from human liver (Asahi Kasei Corporation product number T-73) (1 ) No addition, (2) 1 (W / V)%, (3) 3 (W / V)%, and 2 ml aliquots were lyophilized. Under each condition, a load test at 37 ° C. was performed, and each one was dissolved with 2 ml of distilled water every week until 4 weeks, and ALP activity was measured. The transition of residual activity is shown in Table 1. Significant stabilization was observed in the system to which BSA was added compared to the system to which BSA was not added.
(Example 2)
Add 3% sucrose, galactose, and lactose to 20 mM BES-NaOH buffer (pH 7.5) containing 3% BSA, 0.1 mM zinc chloride, 30 mM valine, 400 U / L human liver-derived ALP, and add 3 ml each. Two vials were dispensed and lyophilized. The freeze-dried product was dissolved by adding 3 ml of distilled water, and ALP activity was measured. The remaining one was placed in a 37 ° C. incubator and left for one week. Thereafter, ALP activity was measured in the same manner. As shown in Table 2, those to which galactose and lactose of the present invention were added had significantly higher residual activity than the sucrose addition system.
(Example 3)
5 (W / V)% galactose in 40 mM MPIPES-NaOH buffer containing 3% BSA, 0.5 mM magnesium chloride, 10 μM zinc chloride, 400 U / L ALP derived from human liver (Asahi Kasei Corporation product number T-73) , Fructose and lactose were added, and 2 ml each was dispensed into vials and lyophilized. The lyophilization was performed at a freezing temperature of −50 ° C. and a vacuum, followed by primary drying at −10 ° C. for 12 hours, secondary drying at 20 ° C. for 24 hours, and vacuum-plugging. 2 ml of distilled water was added to the lyophilized product to dissolve it, and ALP activity was measured for each sugar additive. The remaining vials were placed in a 37 ° C. incubator, dissolved one by one in 2 ml of distilled water every week, and ALP activity was measured up to 3 weeks. The residual activity is shown in Table 3 with the enzyme activity immediately after lyophilization as 100%. Residual activity was significantly higher for any galactose, fructose, or lactose sugar compared to no sugar added.
Example 4
(1) No sugar added to 40 mM BES-NaOH buffer containing 3% BSA, 0.5 mM magnesium chloride, 10 μM zinc chloride, 400 U / L human liver-derived ALP (Asahi Kasei Corporation product number T-73), (2 ) Add galactose 1 (W / V)%, (3) 3 (W / V)%, (4) 5 (W / V)%, (5) Mannose 3 (W / V)% And lyophilized. Under each condition, a load test at 37 ° C. was performed, and each one was dissolved with 2 ml of distilled water every week until 4 weeks, and ALP activity was measured. Table 4 shows the transition of the residual activity. Although the effect of adding galactose was recognized, the activity of mannose decreased rapidly after the third week.
(Example 5)
Galactose 5 (W) in 40 mM BES-NaOH buffer containing 1% BSA, 3% dextran 60K, 0.5 mM magnesium chloride, 10 μM zinc chloride, 400 U / L ALP derived from human liver (Asahi Kasei Corporation product number T-73) / V)% or 3 lactose 5 (W / V)% was added, and 2 ml was dispensed into vials and freeze-dried. After the freeze-dried product was dissolved and the initial activity was measured, four of each were placed in a thermostat at 25 ° C., 37 ° C., and 45 ° C., and a load test was performed. The results are shown in Table 5. Next, based on this result, it was applied to the case of storage at 4 ° C. and −10 ° C. using the Arrhenius equation (J. Biol. Stand., 12, p195-224, 1984). The period until the residual activity reached 98% was calculated and shown in Table 6. In both cases of galactose and lactose, the calculation results were predicted to be stable over a long period of about 15 years when stored at 4 ° C for 1 year or longer and at -10 ° C.
(Example 6)
5 (W / V)% galactose in 40 mM BES-NaOH buffer containing 3% BSA, 0.5 mM magnesium chloride, 10 μM zinc chloride, 400 U / L ALP derived from human liver (Asahi Kasei Corporation product number T-73) Alternatively, lactose was added as a comparative example with trehalose, dispensed in 2 ml vials, and lyophilized. The changes in activity in the refrigerator (5 ° C.) were examined for 6 months and are shown in Table 7. The lyophilized preparation added with trehalose shows a decrease in activity of 8.6% in 4 months, whereas the preparation of the present invention does not show a decrease in activity.
(Example 7)
5 (W / V)% galactose in 40 mM BES-NaOH buffer containing 3% BSA, 0.5 mM magnesium chloride, 10 μM zinc chloride, 400 U / L ALP derived from human liver (Asahi Kasei Corporation product number T-73) , Lactose and fructose were added, and 2 ml aliquots were lyophilized. It was dissolved in 2 ml of distilled water and left at 25 ° C. The transition of ALP activity in the lysate is shown in Table 8 as the residual activity. None of the sugars showed any increase in activity and was stable up to 48 hours.
(Example 8)
3% dextran (molecular weight 60000-90000: Wako Pure Chemical Industries) each in 20mMPOPSO-NaOH buffer (pH 7.5) containing 5% galactos, 0.01 mM zinc chloride, 0.5 mM magnesium chloride, 400 U / l human liver-derived ALP Kogyo Co., Ltd.) or 3% BSA was added, and 2 ml was dispensed into vials and freeze-dried under the same freeze-drying conditions as in Example 3. Under each condition, a load test at 37 ° C. was carried out, and each one was dissolved with 2 ml of distilled water every week until 4 weeks, and ALP activity was measured. The transition of residual activity is shown in Table 9. Although dextran was slightly inferior to BSA, the stabilizing effect was confirmed. In addition, although dextran and BSA were not added, they were placed in the control, but could not be lyophilized well.
Industrial Applicability According to the present invention, as an alkaline phosphatase-containing lyophilized preparation for clinical examination, specifically, a human whose activity after condensate is not increased and which can be stored for a long time A stable lyophilized preparation of alkaline phosphatase can be provided.
Claims (5)
(1) ヒト肝由来アルカリフォスファターゼ、
(2) ヒト臓器非特異的アルカリフォスファターゼ。 Following (1) or (2) the human-derived alkaline phosphatase galactose terpolymer zero, lactose and lyophilized formulations, characterized by freeze-drying in the presence of sugar and albumin or dextran selected from the group consisting of fructose alkaline phosphatase stabilization method derived from human;
(1) Alkaline phosphatase derived from human liver,
(2) Non-human organ alkaline phosphatase.
(1) ヒト肝由来アルカリフォスファターゼ、
(2) ヒト臓器非特異的アルカリフォスファターゼ。 The human-derived alkaline phosphatase of (1) or (2) below incorporates a human cell line, a gene encoding the amino acid of the human-derived enzyme, or a gene converted so that at least a part of the amino acid is deleted or replaced The stabilization method according to claim 1, wherein the method is obtained from the obtained transformant cell ;
(1) Alkaline phosphatase derived from human liver,
(2) Non-human organ alkaline phosphatase .
(1) ヒト肝由来アルカリフォスファターゼ、
(2) ヒト臓器非特異的アルカリフォスファターゼ。The stabilization method according to claim 2, wherein the human-derived alkaline phosphatase transformed cell of the following (1) or (2) is a cell into which a gene encoding an amino acid of human organ non-specific alkaline phosphatase is incorporated .
(1) Alkaline phosphatase derived from human liver,
(2) Non-human organ alkaline phosphatase .
(1) ヒト肝由来アルカリフォスファターゼ、
(2) ヒト臓器非特異的アルカリフォスファターゼ。A stabilized human alkaline phosphatase according to the following (1) or (2) is lyophilized in the presence of a saccharide selected from the group consisting of galactose, lactose and fructose and albumin or dextran Human-derived alkaline phosphatase lyophilized preparation ;
(1) Alkaline phosphatase derived from human liver,
(2) Non-human organ alkaline phosphatase .
(1) ヒト肝由来アルカリフォスファターゼ、
(2) ヒト臓器非特異的アルカリフォスファターゼ。 (1) or (2) human-derived alkaline phosphatase 9-6500 U / L, saccharide 0.5 to 20 (W / V)% selected from the group consisting of galactose, lactose and fructose, and albumin or dextran 0.3 The lyophilized preparation according to claim 4, wherein -7 (W / V)% is used;
(1) Alkaline phosphatase derived from human liver,
(2) Non-human organ alkaline phosphatase .
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US8158152B2 (en) * | 2005-11-18 | 2012-04-17 | Scidose Llc | Lyophilization process and products obtained thereby |
CN102109430B (en) | 2009-12-25 | 2013-11-06 | 深圳迈瑞生物医疗电子股份有限公司 | Nucleated red blood cell simulation particle and blood quality control substance and preparation methods and application thereof |
CN102115737B (en) * | 2009-12-31 | 2015-06-03 | 深圳迈瑞生物医疗电子股份有限公司 | Reagent and method for stabilizing alkaline phosphatase or marker of alkaline phosphatase |
CN102269761A (en) | 2010-06-04 | 2011-12-07 | 深圳迈瑞生物医疗电子股份有限公司 | Synthesis process for alkaline phosphatase conjugate |
CN102269762B (en) | 2010-06-04 | 2014-12-10 | 深圳迈瑞生物医疗电子股份有限公司 | Preparation method of conjugate and relative kit |
JP6155647B2 (en) * | 2012-01-13 | 2017-07-05 | 東洋紡株式会社 | Method for producing fructosyl valyl histidine oxidase preparation |
CN102636639A (en) * | 2012-04-19 | 2012-08-15 | 上海蓝怡科技有限公司 | Diluent of alkaline phosphatase marker |
CN102628863B (en) * | 2012-04-19 | 2016-05-11 | 上海蓝怡科技有限公司 | Mark alkaline phosphatase antigen-antibody dilution |
JP6520153B2 (en) * | 2015-01-29 | 2019-05-29 | 東ソー株式会社 | Method for producing enzyme-linked small molecule |
CN109900900B (en) * | 2019-04-18 | 2022-02-01 | 珠海丽珠试剂股份有限公司 | Diluent and kit suitable for alkaline phosphatase labeled procalcitonin antigen or antibody |
CN112680502A (en) * | 2020-12-07 | 2021-04-20 | 郑州标源生物科技有限公司 | Alkaline phosphatase quality control substance and preparation method thereof |
CN118086244A (en) * | 2023-08-18 | 2024-05-28 | 北京中检葆泰生物技术有限公司 | Preparation method of milk-source alkaline phosphatase standard substance |
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